Calcium flux agonists and methods thereof

ABSTRACT

Calcium flux agonists are used to enhance a TLR- or NOD-mediated stimulus and to so increase an immune response of a host and reduce healing time. Preferred calcium flux agonists include Ca 2+  ionophores and SERCA inhibitors and are used in synergistic quantities where a ligand to a TLR or NOD receptor is present.

This application claims priority to our provisional application withSer. No. 61/725,881, filed 13 Nov. 2012, and which is incorporated byreference herein.

FIELD OF THE INVENTION

The field of the invention is compositions and methods of pharmaceuticalcompounds, and especially of topically applied calcium flux agonists.

BACKGROUND OF THE INVENTION

Various calcium flux agonists are known in the art and have vastlydifferent origins. For example, certain compounds act as ionophores andtypically raise intracellular calcium levels by importing calcium ionsinto the cell, while other compounds raise intracellular calcium levelsby increasing calcium secretion from intracellular stores like theendoplasmic reticulum and mitochondria.

Among various other known ionophores, calcimycin (A23187) and ionomycinare natural products (from the Gram+ bacteria Streptomyceschartreusensis and Streptomyces conglobatus, respectively) which wereinitially described as antibiotics decades ago. More specifically, bothA23187 and ionomycin demonstrate direct antibiotic activity against avariety of potential microbial pathogens as was reported in U.S. Pat.No. 3,960,667 and U.S. Pat. No. 3,873,693. Unlike classical antibiotics(e.g., penicillin or tetracycline) that inhibit biochemical pathwaysspecific to microbial proliferation such as bacterial cell wallsynthesis or bacterial ribosome function, A23187 and ionomycin belong toa separate class of antibiotic compounds that bind divalent cations assubstrates with relatively high specificity. For example, A23187 bindingaffinity is characterized by Mn²⁺>Ca²⁺═Mg²⁺>Sr²⁺>Ba²⁺ while ionomycin ischaracterized by Ca²⁺>Mg²⁺>Ba²⁺>Sr²⁺.

On the other hand, thapsigargin is a typical ER secretagogue and can becharacterized as a sesquiterpene lactone. Thapsigargin is isolated froma plant (Thapsia garganica) and acts as a non-competitive inhibitor ofvarious sarco/endoplasmic reticulum Ca2+ ATPases (SERCA).Thapsigargin:SERCA binding demonstrates an affinity constant in the lowpicomolar range and is toxic to both dividing and non-dividing cells. Inanimals, limited skin contact with thapsigargin can result ininflammation and chronic repetitive topical exposure can result innon-malignant papilloma formation when used in conjunction with a strongDNA damaging agent. In addition to thapsigargin (and structural analogslike thapsigargicin, etc.), other SERCA inhibitors include cyclopiazonicacid (CPA) and 2,5-di-tert-butylhydroquinone (DBHQ).

Difficult-to-treat skin infections represent an emerging public healthconcern for several reasons including the ever-increasing number ofdiabetic patients suffering from chronic skin ulcers, the presence ofantibiotic resistant microbial flora (e.g., methicillin-resistantStaphylococcus aureus or MRSA), and the increasing frequency ofoutbreaks of necrotizing fasciitis (or “flesh-eating bacteria disease”).In addition to bacteria, many fungi and viruses can also causesignificant infections of the skin. Although antibiotics remain the besttreatment option for many of these disorders, it would be desirable tostimulate a patient's own cells and their associated functions tofurther improve patient recovery from ongoing infections and possiblyeven to stimulate long-term immunity against the offending organism tolimit pathogenesis upon subsequent encounter.

In higher organisms, epithelial tissues including the skin serve as acritical barrier against pathogen-based, chemical, and physical insults.The epidermal layer of skin is comprised of keratinocytes, immune cellssuch as Langerhans cells and CD8⁺ T cells, Merkel cells and melanocytes.In addition to Langerhans cells, which are a subtype of dendritic cells(DC) responsible for disease surveillance, keratinocytes (which accountfor ˜95% of the total epidermal population) also serve as immunesentinels through their expression of various pattern recognitionreceptors such as members of the toll-like receptor (TLR) proteins,C-type lectin receptors (CLR), inflammasomes, etc.

Activation of these receptors in keratinocytes by their cognate ligandsresults in the release of both chemokines such as interleukin-8 (IL-8),CCL2, and CCL20 to recruit other immune cells as well asimmune-regulating cytokines such as TGF-β and IL-10. Below theepidermis, the dermis is comprised of a larger variety of cell typesincluding various subsets of CD4⁺ (Th1, Th2, Th17, etc) andnon-classical (e.g. γδ, NK-, etc.) T lymphocytes, various antigenpresenting cells (such as macrophages and dermal and plasmacytoid DC),mast cells and fibroblasts many of which express the various patternrecognition proteins described above. As such, both epidermal and dermalcells cooperate to prevent microbial invasion or other physical insultsthat could lead to significant disease.

The TLR proteins (TLR 1-10 in man) are type I membrane proteins whichserve as pattern recognition receptors (PRR) for specific classes ofligands associated with disease and tissue homeostasis and as such areexpressed on a variety of immune and non-immune cell types alike. Aswith all receptors, TLR signal transduction is triggered by ligandbinding, and ligands may be grouped into pathogen-associated molecularpatterns (or PAMP) and disease-associated molecular patterns (DAMP).

TLR-recognized PAMP include bacterial lipoproteins/lipopeptides,liposaccharides, flagellin, and unmethylated CpG DNA, fungal cell wallcomponents (e.g. zymosan), and viral nucleic acids (dsRNA, ssRNA, andCpG DNA), while DAMPS are derived directly from the host, can occur inthe absence of infection, and are recognized predominantly by only twomembers of the TLR family of proteins (either TLR2 and/or TLR4).

Once activated by their appropriate ligands, TLR initiate signalingcascades which serve both to limit the extent of infection/disease andto trigger tissue repair. With regards to the latter, TLR ligandrecognition results in the up-regulation of antimicrobial activity andcauses the activation/maturation of various immunological players tocomplete the destruction of the invading pathogen. For example, TLRactivation can result in the release of reactive oxygen species (ROS),antimicrobial peptides, and upregulation of phagocytic function ininnate cells such as macrophages, neutrophils, keratinocytes, etc. Inaddition to their role in combating disease, TLRs are also involved intissue repair and regeneration as demonstrated in various models oftissue damage (including those induced by chemical, radiation, surgical,and infectious injury).

A further class of pattern recognition receptors is formed by theNOD-like receptor protein family, and includes NOD1 and NOD2 as the mostprominent members. NOD1 and NOD2 are intracellular pattern recognitionreceptors, which are similar in structure to resistance proteins ofplants, and mediate innate and acquired immunity by recognizingbacterial molecules containing D-glutamyl-meso-diaminopimelic acid andmuramyl dipeptide, respectively. Following stimulation by theirrespective ligands, both NOD proteins interact with RIPK2 throughrespective recognition domains, which ultimately results in activationof the transcription factor NF-κB.

Previous efforts to characterize the response of intact skin to topicalapplication of small molecule agonists of signal transduction (such asthe protein kinase C agonist TPA/PMA or sustained calcium flux agonistslike A23187, ionomycin, or thapsigargin) demonstrated a spectrum ofdownstream results. For example, topical application of the phorbolester TPA caused vasodilation, microvascular permeability alterations,inflammatory cell recruitment, and the release of pro-inflammatoryfactors from various cell types. In contrast to PKC agonists, topicaltreatment with sustained calcium flux agonists (SCFA) resulted in skininflammation and hyper-proliferation. On the other hand, exposure ofcells to the TLR ligand (LPS) in the presence of relatively highquantities of a calcium ionophore (ionomycin) did not lead to anymeasurable immunostimulatory effect (Proc Natl Acad Sci USA. 2012 Jul.10; 109(28): 11282-7). When applied individually at relatively highdosages, calcium ionophores and TLR ligands are known to stimulatedifferentiation or to activate dendritic cells as discussed in US2012/0272700A1 and US 2013/0183343A1.

Therefore, while numerous compositions and uses for calcium fluxagonists and/or ligands for TLR/NOD are known in the art, there is stilla need to provide compositions and methods that provide improvedimmunomodulatory activity.

SUMMARY OF THE INVENTION

The present inventive subject matter is drawn to various compositionsand methods of calcium flux agonists in which these compounds are usedto modulate an immune response to a TLR- or NOD-mediated event, andespecially to synergistically increase responses to TLR and/or NODligand binding. Notably, synergistic effect with respect to immunestimulation is observed where the calcium flux agonist is present insuboptimal concentrations.

Viewed from a different perspective, the inventors contemplate use ofcalcium flux agonists to amplify intracellular Ca²⁺ dependent TLR/NODsignaling. Therefore, and among other suitable uses, especiallycontemplated uses include pre-conditioning of tissue to allow for anenhanced response to a TLR/NOD-dependent stimulus (e.g., infection orinjury), and/or treatment of topical or other infections of theepithelium with calcium flux agonists to amplify intracellular Ca²⁺dependent TLR/NOD signaling. Consequently, the inventors alsocontemplate topical pharmaceutical and cosmetic compositions forprevention and/or treatment of skin infections and other conditions(e.g., diabetic ulcers) to stimulate wound healing, and/or to decreasescarring.

In one aspect of the inventive subject matter, the inventors contemplateuse of a calcium flux agonist to enhance an immune response of an immunecompetent cell to a ligand of a pattern recognition receptor. Forexample, where the calcium flux agonist is a calcium ionophore,preferred agonists include ionomycin, calcimycin, beauvericin, calciumionophore II, calcium ionophore IV, calcium ionophore V, and calciumionophore VI, and where the agonist is a SERCA inhibitor, preferredSERCA inhibitors include DBHQ (2,5-di-tert-butylhydroquinone),thapsigargin, ruthenium red, gingerol, paxilline, and cyclopiazonicacid. Among other phenomena observable, the enhanced immune response istypically evidenced by an increased IL-8 secretion and/or an increasedactivation of NF-κB signaling, and it is preferred that the immuneresponse is synergistically enhanced by the calcium flux agonist in thepresence of the ligand, particularly where the calcium flux agonist isused at a suboptimal concentration (with respect to a maximum effect ofthe calcium flux agonist in the absence of the ligand).

With respect to suitable cells it is generally contemplated that thecells are immune competent cell, which will preferably reside in theepidermal or dermal layer of skin. Moreover, the immune competent cellswill generally express a TLR receptor and/or a NOD receptor as thepattern recognition receptor. Thus, ligands will typically include PAMPand DAMP ligands.

Consequently, the inventors also contemplate use of a calcium fluxagonist in the manufacture of a topically applied medicament to enhancean immune response in skin. In such uses, the immune response isgenerally associated with binding of a (PAMP or DAMP) ligand to apattern recognition receptor in an immune competent cell, and thepattern recognition receptor is most typically a TLR receptor or a NODreceptor. As noted above, preferred calcium flux agonists includecalcium ionophores (e.g., ionomycin, calcimycin, calcium ionophore II,calcium ionophore IV, calcium ionophore V, or calcium ionophore VI), andSERCA inhibitors (e.g., 2,5-di-tert-butylhydroquinone, thapsigargin,ruthenium red, gingerol, paxilline, or cyclopiazonic acid, etc.), and/orthe calcium flux agonist is present in the medicament at a concentrationsuch that the agonist is present in the cell in the presence of theligand at a suboptimal concentration.

Consequently, and viewed from a different perspective, the inventorsalso contemplate use of a calcium flux agonist in the manufacture of atopically applied medicament to enhance wound healing of skin. In suchuses, the calcium flux agonist is typically present in an amounteffective to activate NF-κB signaling of cells in the wound. Such usesare particularly advantageous where the wound is infected with abacterial pathogen (typically expressing or producing a ligand for a TLRreceptor and/or a NOD receptor).

In yet another aspect of the inventive subject matter, the inventorsalso contemplate a pharmaceutical composition that comprises a calciumflux agonist (e.g., calcium ionophore or a SERCA inhibitor) in apharmaceutically acceptable carrier, wherein the pharmaceuticalcomposition is formulated for topical application to injured or infectedskin, and wherein the calcium flux agonist is present in an amount thatenhances, upon application of the formulation to the injured or infectedskin, an immune response of an immune competent cell in the injured orinfected skin to a ligand of a pattern recognition receptor (e.g., TLRreceptor or NOD receptor). While such pharmaceutical compositions may beformulated, for example, as a liquid, a spray, or a gel, it is alsocontemplated that the pharmaceutical composition is formulated fortopical application to injured or infected skin via a solid carrier thatis applied to the injured or infected skin (e.g., wound dressing or bandaid impregnated with the pharmaceutical composition). For example, theskin may be infected with a (e.g., bacterial) pathogen that comprises aligand for a TLR receptor or a NOD receptor.

In particularly preferred pharmaceutical compositions, the amount of thecalcium flux agonist (e.g., ionomycin, calcimycin, or thapsigargin)synergistically enhances the immune response in the presence of theligand as compared to the immune response in the absence of the ligand.

Therefore, the inventors also contemplate a method of enhancing animmune response of a cell expressing a pattern recognition receptor(e.g., TLR receptor or a NOD receptor) to a ligand (e.g., PAMP) of thepattern recognition receptor, wherein the method includes a step ofexposing the cell in the presence of the ligand to a calcium fluxagonist (e.g., SERCA inhibitor or calcium ionophore) in an amount thatenhances the immune response.

Most typically, the immune response is evidenced as an increased IL-8secretion and/or an increased activation of NF-κB signaling, and it isespecially preferred that the ligand and the calcium flux agonist arepresent in synergistic quantities. While not limiting to the inventivesubject matter, it is further preferred that the cell is located in adermal layer or epidermal layer of skin (e.g., injured or infectedskin).

Viewed from a different perspective, the inventors therefore alsocontemplate a method of treating injured or infected skin in which inone step the injured or infected skin is contacted with a calcium fluxagonist (e.g., calcium ionophore or a SERCA inhibitor) in an amount thatenhances an immune response (e.g., increased IL-8 secretion or increasedactivation of NF-κB signaling) and that increases wound healing.

Thus, the inventors also contemplate a method of modulating an immuneresponse to a TLR- or NOD-mediated stimulus in a tissue (e.g.,epithelial tissue) or cell, in which in one step the tissue or cell iscontacted (e.g., topically applied) with a calcium flux agonist at aconcentration effective to increase an intracellular calciumconcentration as compared to an intracellular calcium concentrationwithout the calcium flux agonist. Most typically, the concentration ofthe calcium flux agonist (e.g., calcium ionophore or SERCA inhibitor) iseffective to modulate the immune response to the TLR-mediated stimulus.In some aspects of the inventive subject matter, the TLR- orNOD-mediated stimulus is a bacterial, viral, or fungal infection, whilein other aspects the TLR- or NOD-mediated stimulus is a tissue injury.

Various objects, features, aspects and advantages of the inventivesubject matter will become more apparent from the following detaileddescription of preferred embodiments, along with the accompanyingdrawing figures in which like numerals represent like components.

BRIEF DESCRIPTION OF THE DRAWING

FIGS. 1A-1C are graphs depicting the dose response curves for variouscalcium flux agonists (1A: A23187; 1B: Ionomycin; 1C: Thapsigargin) withrespect to strength of NF-κB signaling and IL-8 production.

FIG. 2 is an exemplary graph depicting synergistic effect of variouscalcium flux agonists with an exemplary TLR ligand (Pam2CYS).

FIGS. 3A-3E show various graphs illustrating the synergistic effect ofexemplary calcium flux agonists (FIG. 3A: A23187; FIG. 3B: Ionomycin;FIG. 3C: Cyclopiazonic acid; FIG. 3D: DBHQ; FIG. 3E: Thapsigargin) andexemplary TLR and NOD ligands for respective TLR and NOD receptors withrespect to strength of IL-8 production.

FIG. 4 shows graphs illustrating the effect of different Ca²⁺ modulatingcompounds on the synergistic action with respect to NF-κB activation.

FIGS. 5A-5E show graphs depicting the effect of live bacterial growth onκB-LUC THP-1 reporter cells (FIG. 5A), augmentation of NF-κB response bythapsigargin (FIG. 5B), and the extracellular requirement of Ca²⁺ forcalcium flux agonist signal amplification (FIGS. 5C-5E).

FIG. 6 shows graphs illustrating the requirement of human primarymonocytes and granulocytes for extracellular Ca²⁺ for optimal killing ofphagocytosed S. aureus.

FIG. 7 shows photographs of wound healing using selected calcium fluxagonists.

FIG. 8 shows graphs indicating wound size and bacterial burden followingwound treatment with control and selected calcium flux agonists.

FIGS. 9A-9C show graphs for antibiotic sensitivity of S. aureus againstA23187, ionomycin, and CPA, respectively.

FIG. 10 is a graph depicting lack of direct antibiotic effect ofthapsigargin against S. aureus.

FIG. 11 is a graph showing improved killing of intracellular S. aureusby thapsigargin-treated cells.

DETAILED DESCRIPTION

The inventors have discovered that calcium flux agonists that increaseintracellular free Ca²⁺ concentration (especially calcium ionophores andSERCA inhibitors) can be effectively used to modulate and/or enhance theimmune response of a host to a TLR- or NOD-mediated event. In aparticularly notable aspect, calcium flux agonists synergisticallyincreased host responses to TLR and/or NOD ligand binding where thecalcium flux agonist is present in a substantial suboptimalconcentration.

Indeed, the inventors have discovered as further detailed below thatinnate immune cells help to control infection by recognizing S. aureusbacteria or their shed products (and other pathogens and pathogenfragments) in a Ca²⁺-dependent manner to become activated, which canthen be effectively augmented by exposure of the affected cells tosuboptimal doses of Ca²⁺+ flux agonists. Since many common pathogen(e.g., S. aureus) products are predominantly recognized by TLR and NODreceptors, the inventors investigated and also confirmed that pathogenproducts are indeed activating and that such activation can be furthersignificantly enhanced using numerous Ca²⁺ flux agonists (which can beabrogated or substantially reduced by calcium chelating agents).Moreover, the inventors also demonstrated that Ca²⁺ is a critical factorin human monocytes and granulocytes (especially neutrophils) foreffective killing of phagocytosed bacteria. Such findings also directlytranslated into a mammalian (murine) model of skin infection and woundhealing following live infection.

In one aspect of the inventive subject matter, the inventors thereforecontemplate various compositions and methods for (typically topical)treatment and/or prophylaxis that are effective in stimulating the hostcells' capability of immune response to a pathogen and that areeffective in reducing scarring and time-to-closure of a topical wound,especially where the wound is infected with one or more pathogens thatexpress or otherwise comprise a ligand for TLR and/or NOD. For example,the inventors contemplate topical application of a SERCA inhibitor(e.g., thapsigargin) and/or an ionophore (e.g., ionomycin and/or A23187(calcimycin) to prevent or treat a superficial skin infection (e.g.,bacterial infection).

In that context, the inventors discovered that topical application ofSERCA inhibitors and/or ionophores greatly decreased bacterial burden insuperficial skin infections (e.g., S. aureus) with concomitantimprovement in wound healing kinetics in a live animal infection model.Remarkably, the antibacterial effect was not (in the case of the SERCAinhibitor thapsigargin) or not entirely (in the case of ionophores)attributable to a direct antibiotic effect where the calcium fluxagonist acted as a biocide against the pathogen, but rather to the roleof the calcium flux agonists as an immunological adjuvant. While notwishing to be bound by any theory or hypothesis, the inventorscontemplate that the antibacterial effect may be due to synergisticactivation of cytokine release and activation of the nuclear factor-κB(NF-κB) signaling pathway when calcium flux agonists were provided inthe presence of TLR or NOD ligands.

Even more remarkable, very strong synergy between TLR and/or NODactivation and calcium flux agonists (ionophores/SERCA inhibitors) wasobserved for IL-8 production and activation of NF-κB signaling. Forexample, human promonocytic THP-1 cells were incubated in the presenceor absence of a suboptimal dose of the TLR2 ligand Pam2CSK4 and comparedto similarly treated cells which also received suboptimal doses ofionophore (here: A23187 and ionomycin). Interestingly and as furthershown in more detail below, the inventors found that both agonistssignificantly increased the amount of IL-8 produced beyond the levelsthat would be produced if the individual responses were cumulative, thusindicating true synergy. In another example, κB-LUC THP-1 cells (aTHP-1-derived transfectant line harboring an NF-κB-driven luciferaseexpression cassette) were cultivated in the presence or absence of asuboptimal dose of the TLR2 ligand Pam2CSK4 and compared to similarlytreated cells that also received suboptimal doses of various SERCAinhibitors. Notably, and as also shown in more detail below, theinventors found that all of the tested agonists (here: thapsigargin,cyclopiazonic acid) significantly increased the amount of luciferaseproduced beyond levels that would be produced if individual responseswere cumulative and so once more indicate synergy.

In that context it should be noted that NF-κB impacts adaptive immunitythrough its involvement in mediating cellular activation, inflammatorycytokine secretion, proliferation, and survival, while IL-8 is apowerful chemo-attractant for immune cells like neutrophils, an innateimmune cell type critical for antibacterial and antifungal responses.Interestingly, ligand activation of TLR4 in the presence of the Ca²⁺flux inducing agent ionomycin results in the synergistic production ofarachidonic acid-derived eicosanoid lipids in a mouse macrophage line,indicating the possibility of combining Ca²⁺ flux inducing agents withTLR ligands for preventative and curative effects.

The inventors therefore contemplate in one aspect of the inventivesubject matter that ionophores (e.g., A23187 and/or ionomycin) willproduce a synergistic effect in vivo with infection/disease associatedTLR and/or NOD ligands to alter in situ immune cell response and repairprocesses. Such effects are readily ascertained as the inventorsdemonstrated in more detail below by use of topically-appliedformulations containing various ionophores to alter the course ofsuperficial skin infection, for example, induced by S. aureus as a modelsystem, using bacterial burden and wound size as indicators ofprophylactic and/or therapeutic success.

Similarly, the inventors also contemplate in another aspect of theinventive subject matter that thapsigargin (and various other SERCAinhibitors) produce a synergistic effect in vivo with infection/diseaseassociated TLR and/or NOD ligands to alter in situ immune cell andrepair processes in vivo. As noted above, such effects are readilyascertained as the inventors demonstrated in more detail below by use oftopically-applied formulations that contain various SERCA inhibitors(and especially thapsigargin) to alter the course of superficial skininfection, for example, induced by S. aureus as a model system, usingbacterial burden and wound size as indicators of prophylactic and/ortherapeutic success.

Therefore, it should be appreciated that the inventors especiallycontemplate use of a calcium flux agonist to enhance the immune responseof one or more immune competent cells to a ligand of a patternrecognition receptor. Most typically, suitable immune competent cellsare cells that are part of the cellular immune system (e.g., a B-cell, aT-cell, an antigen-presenting cell or innate sentinel cell, andespecially dendritic cells, macrophages, mast cells, monocytes, etc.),and it is generally preferred that such immune competent cells are inthe dermal or epidermal layer of skin. Consequently, the inventors alsocontemplate the use of a calcium flux agonist to manufacture a topicallyapplied medicament to enhance an immune response and/or wound healing(e.g., reduce time-to-closure) in skin. In such uses, it is generallycontemplated that the immune response is associated with binding of aligand to a pattern recognition receptor in an immune competent cell.Among other pattern recognition receptors, especially suitable patternrecognition receptors include those of the TLR and NOD families, andespecially TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10,TLR11, TLR12, and TLR13, as well as NOD1, NOD2, NOD3, NOD4, NOD5, andCIITA. Therefore, it should be appreciated that the ligand may varyconsiderably, and contemplated ligands include all ligands known to bindto the TLR and/or NOD receptors, and most preferably pathogen-associatedmolecular pattern ligands (PAMP) and damage/disease-associated molecularpattern ligands (DAMP). Consequently, suitable PAMP ligands will includevarious lipopeptides, glycolipids, lipoteichoic acid, heat shockproteins, beta-glucans, fibrinogen, heparin sulfate fragments,hyaluronic acid fragments, RNA (and esp. ssRNA), DNA (and csp. CpGsequences), profiling, etc. Likewise, suitable DAMPs will includevarious nuclear and/or cytosolic proteins, and especially heat shockproteins, HMGB1, DNA, RNA, and fragments of proteins derived fromextracellular matrix.

In yet another aspect of the inventive subject matter, the inventorsalso contemplate a method of enhancing an immune response of a cell(that expresses one or more of the pattern recognition receptors notedabove) to a ligand of the pattern recognition receptor. As noted above,the nature of the ligand will predominantly depend on the type ofreceptor, and all receptors and ligands as discussed above are suitablefor contemplated methods. In especially preferred methods, the cell isexposed in the presence of the ligand to a calcium flux agonist in anamount that enhances the immune response. Of course, it should be notedthat the ligand may be delivered (e.g., together with the calcium fluxagonist) as part of a treatment regimen. However, and more typically,the ligand will be provided by a pathogen that is present in or near thecell, or provided by the host as a DAMP. While not limiting to theinventive subject matter, the enhanced immune response will typically becharacterized by at least one of an increased IL-8 production and anincreased NF-κB signaling (e.g., increased expression of a gene underthe control of NF-κB in the presence of the calcium flux agonist ascompared to the expression of the same gene in the absence of thecalcium flux agonist). As is readily evident from the experimentaldetails below, the enhanced immune response is typically a synergisticincrease.

Therefore, the inventors also contemplate a method of (prophylactic)treating injured or infected skin by contacting the injured or infectedskin with a calcium flux agonist in an amount that enhances an immuneresponse and that increases wound healing as further shown in moredetail below. Viewed from a different perspective, it should thus beappreciated that one or more calcium flux agonists can be employed tomodulate an immune response to a TLR- or NOD-mediated stimulus in atissue or cell.

Contemplated Calcium Flux Agonists

Compounds contemplated suitable for use herein are generally deemed tobe useful for prophylaxis and/or treatment of various infectiousdiseases and trauma, and especially with infectious disease of the skinor other epithelium, particularly where the host response to the diseaseand/or trauma is associated with a TLR or NOD response pathway.Therefore, it is generally preferred that the compounds according to theinventive subject matter will be calcium flux mediators (and especiallyagonists) that lead to an at least temporary, and more typicallysustained increase of intracellular calcium.

Therefore, especially preferred compounds include ionophores and SERCAinhibitors well known in the art. Most preferably, suitable ionophoresare calcium ionophores, and especially ionomycin, calcimycin, calciumionophore II, calcium ionophore IV, calcium ionophore V, or calciumionophore VI, while particularly preferred SERCA inhibitors include2,5-di-tert-butylhydroquinone (DBHQ), thapsigargin, ruthenium red,gingerol, paxilline, or cyclopiazonic acid. It should also be noted thatwhile the above is a list of preferred compounds, the list is notexhaustive, and individual compounds may be combined to form a mixtureof two or more calcium flux agonists (e.g., to provide an extracellularand intracellular agonist), and/or additional compounds may be added.

Of course, it should be appreciated that (where appropriate)contemplated compounds may have one or more asymmetric centers or groupsthat may give rise to isomeric, tautomeric, or other steric isoforms(e.g., R-, and/or S-configuration, E/Z configuration, tautomericisoforms, enantiomers, diastereomers, etc.), and each of such forms andmixtures thereof are expressly contemplated herein. Additionally, itshould be appreciated that contemplated calcium flux agonists may bechemically modified to achieve a desired physicochemical parameter(e.g., solubility in aqueous solvents, membrane permeability,selectivity towards Ca²⁺, etc.) Therefore, suitable calcium fluxagonists may be fully synthetic, semi-synthetic, or isolated from hoststrains producing such ionophores.

Moreover, contemplated compounds may also be converted to prodrugs toincrease delivery and/or target specificity to an affected tissue ororgan. The term “prodrug” as used herein refers to a modification ofcontemplated compounds, wherein the modified compound exhibits lesspharmacological activity (as compared to the unmodified compound) andwherein the modified compound is converted within a target cell ortarget organ back into the unmodified form. For example, conversion ofcontemplated compounds into prodrugs may be useful where the active drugis too toxic for safe systemic administration, or where the contemplatedcompound is poorly absorbed by the digestive tract, or where the bodybreaks down the contemplated compound before reaching its target. Thereare numerous methods for the preparation of prodrugs known in the art,and all of those are contemplated herein. For example, suitable prodrugapproaches are described in Prodrugs (Drugs and the PharmaceuticalSciences: a Series of Textbooks and Monographs) by Kenneth B. Sloan(ISBN: 0824786297), or in Hydrolysis in Drug and Prodrug Metabolism:Chemistry, Biochemistry, and Enzymology by Bernard Testa (ISBN:390639025X), which are to the appropriate extent incorporated byreference herein.

Similarly, it should be noted that contemplated compounds may also beless active in the form as described herein, and be more active asmetabolite or metabolites that are formed in vivo. For example,contemplated compounds may be transformed by the hepatic phase I and/orphase II enzyme system, or by gastric acidity, intestinal microbialenvironment, or other biochemical process. Thus, suitable compounds maybe oxidized, hydroxylated, ligated to a carbohydrate, etc.

Contemplated Compositions

It is generally contemplated that contemplated calcium flux agonists areprovided in a composition that is suitable for delivery to a cell ortissue. Therefore, suitable compositions will include liquidcompositions, gels, solid compositions, all of which may be associatedor coupled to a carrier, or applied directly to the cell or tissue. Mosttypically, such compositions will include an aqueous solvent orotherwise pharmaceutically acceptable carrier together with one or morecalcium flux agonists. In particularly preferred aspects of theinventive subject matter, the pharmaceutical composition is formulatedfor topical application to an injured or infected tissue, morepreferably epithelial tissue, and most preferably skin. Moreover, it isgenerally preferred that the calcium flux agonist is present in anamount that enhances, upon application of the formulation to the cell ortissue, an immune response of an immune competent cell in the injured orinfected cell or tissue to one or more ligands of a pattern recognitionreceptor (typically TLR and/or NOD receptor). It is still furtherpreferred that the calcium flux agonist is present in an amount thatsynergistically enhances the immune response in the presence of theligand as compared to the immune response in the absence of the ligand.

Therefore, it should be appreciated that the compositions according tothe inventive subject matter may be administered using various routes,including topically, nasally, by inhalation, orally, parenterally, etc.wherein the term “parenteral” as used herein includes subcutaneous,intravenous, intramuscular, intraarticular, intrasynovial, intrathecal,intrahepatic, intralesional, and intracranial administration (typicallyinjection or infusion). Most preferably, however, the compositions areadministered topically in a liquid, gel, or solid form.

For example, the pharmaceutical compositions of this invention may beadministered topically to areas or organs readily accessible by topicalapplication, including the eye, the skin, the lower intestinal tract, orareas exposed during surgical intervention. There are numerous topicalformulations known in the art, and all of such formulations are deemedsuitable for use herein.

For example, contemplated compositions may be formulated in a suitableointment containing the active component suspended or dissolved in oneor more carriers. Carriers for topical administration of the compoundsof this invention include mineral oil, liquid petrolatum, whitepetrolatum, propylene glycol, polyoxyethylene, polyoxypropylenecompound, emulsifying wax and water. Alternatively, the pharmaceuticalcompositions can be formulated in a suitable lotion or cream containingthe active components suspended or dissolved in one or morepharmaceutically acceptable carriers. Suitable carriers include mineraloil, sorbitan monostearate, polysorbate 60, cetyl esters wax, cetearylalcohol, 2-octyldodecanol, benzyl alcohol and water. As at least some ofthe active compounds are highly hydrophobic, it is contemplated that theformulation will take into account relatively poor solubility and thusmay be prepared as an emulsion, as nanovesicular particles, oradministered in a hydrophobic base under occlusion.

Alternatively, contemplated formulations may also be injected into skinor other site of administration. Most preferably, sterile injectableforms of contemplated compounds will include emulsions, aqueoussolutions, or oleaginous suspensions. These suspensions may beformulated according to techniques known in the art using suitabledispersing or wetting agents and suspending agents. The sterileinjectable preparation may also be prepared as a sterile injectablesolution or suspension in a non-toxic parenterally acceptable diluent orsolvent, for example as a solution in 1,3-butanediol. Among otheracceptable vehicles and solvents, especially contemplated liquidsinclude water, Ringer's solution, and isotonic sodium chloride solution.In addition, sterile, fixed oils may be employed as a co-solvent orsuspending medium (e.g., natural or synthetic mono- or diglycerides).Fatty acids may also be used, and suitable fatty acids include oleicacid and its glyceride derivatives, olive oil, castor oil, especially intheir polyoxyethylated versions. Such oil solutions or suspensions mayfurther contain a long-chain alcohol diluent or dispersant.

In another example, contemplated compounds may be orally administered inany orally acceptable dosage form, including capsules, tablets, aqueoussuspensions, or solutions. In the case of tablets for oral use, allpharmaceutically acceptable carriers (e.g., lactose, corn starch, etc)are deemed suitable. Similarly, various lubricating agents may be added(e.g., magnesium stearate). For oral administration in a capsule form,useful diluents include lactose and dried corn starch.

With respect to the amount of contemplated compounds in the composition,it should be recognized that the particular quantity will typicallydepend on the specific formulation, active ingredient, and desiredpurpose. Therefore, it should be recognized that the amount ofcontemplated compounds will vary significantly. However, it is generallypreferred that the compounds are present in a minimum amount effectiveto deliver prophylactic and/or therapeutic effect in vitro and/or invivo. Viewed from another perspective, the calcium flux agonist istypically present in an amount effective to activate NF-κB signalingand/or increase IL-8 production of cells in vitro, or in a wound orotherwise diseased (e.g., infected) tissue.

Moreover, it is generally preferred that the calcium flux agonist isprovided to the cell or tissue such that the agonist will be present inthe cell (in the presence of the TLR or NOD ligand) at a suboptimalconcentration with respect to a maximum effect of the calcium fluxagonist in the absence of the ligand. In this context, it should benoted that the suboptimal concentration of the calcium flux agonist is aconcentration that is well below the maximum response (with respect toNF-κB and/or IL-8 production) obtainable with the calcium flux agonist.Thus, sub-optimal concentration of the calcium flux agonist will beconcentration that will provide equal or less than 80%, equal or lessthan 70%, equal or less than 60%, between 20-60%, or between 10-50% ofthe dosage that provides a maximum effect (with respect to NF-κB and/orIL-8 production) for that calcium flux agonist. For example, as can beseen from the experimental data below, an exemplary suboptimalconcentration for thapsigargin is between 10-50 nM (e.g., about 20 nM),which is well below a maximum effect obtainable for thapsigargin as canbe seen from FIG. 1C. Likewise, an exemplary suboptimal concentrationfor A23187 is between 100-500 nM (e.g., about 316 nM), which is wellbelow a maximum effect obtainable for A23187 as can be seen from FIG.1A, and an exemplary suboptimal concentration for ionotnycin is between300 nM-5 μM (e.g., about 1 μM), which is well below a maximum effectobtainable for ionomycin as can be seen from FIG. 1B. Viewed fromanother perspective, suboptimal concentrations will therefore becharacterized as concentrations below which an acute toxic effect can beobserved for a cell or tissue exposed to the calcium flux agonist.

As is shown in more detail below, maximum response and suboptimalconcentrations can be readily determined using an IL-8 ELISA test and/ora luminescence test. In preferred aspects of the inventive subjectmatter, the suboptimal concentration will be a concentration at whichthe response is equal or less than 70% of the maximum response, moretypically equal or less than 50% of the maximum response, and mosttypically equal or less than 30% of the maximum response. Thus,suboptimal concentrations will be in the range of between 1-20% of themaximum response, between 20-40% of the maximum response, between 40-60%of the maximum response, between 60-80% of the maximum response, orbetween 80-95% of the maximum response. Likewise, it is noted that theligand may also be present in a suboptimal concentration, and thesuboptimal concentration will be a concentration at which the responseto the ligand is equal or less than 70% of the maximum response, moretypically equal or less than 50% of the maximum response, and mosttypically equal or less than 30% of the maximum response for the ligandalone. Thus, suboptimal concentrations will be in the range of between1-20% to 1-40% of the maximum response, between 20-40% to 20-80% of themaximum response, between 30-70% of the maximum response, between 40-80%of the maximum response, or between 50-95% of the maximum response forthe ligand alone.

Consequently, in at least some embodiments, contemplated compounds arepresent in an amount of between about 0.1 ng/ml to about 100 mg/ml, moretypically in an amount of between about 10 ng/ml to about 10 mg/ml, andmost typically between about 1 μg/ml to about 100 μg/ml. Where theformulation is a solid or a gel, contemplated compounds will be presentin an amount of between about 0.1 ng/g to about 100 mg/g, more typicallyin an amount of between about 10 ng/g to about 10 mg/g, and mosttypically between about 1 μg/g to about 100 μg/g. Viewed from adifferent perspective, the calcium flux agonist will typically bepresent in the formulation at a concentration of between 0.1 μM to 10μM, between 10 μM to 100 μM, between 100 μM to 1 mM, between 1 mM to 10mM, or between 10 mM to 100 mM. Additionally, and with respect to theconcentration of the calcium flux agonist at the cell or tissue(“effective exposure concentration”), it is generally preferred that theeffective exposure concentration will be between 1 μM and 1 mM, and mostpreferably at a suboptimal concentration for the respective calcium fluxagonist. Therefore, thapsigargin will typically have an effectiveexposure concentration of between 1 nM to 1 μM, more typically between 1nM to 500 nM, and most typically between 1 nM to 50 nM, while DBHQ andCPA will typically have an effective exposure concentration of between100 nM to 500 μM, more typically between 500 nM to 100 μM, and mosttypically between 1 μM to 50 μM. On the other hand, ionomycin and A23187will typically have an effective exposure concentration of between 10 nMto 100 μM, more typically between 100 nM to 50 μM, and most typicallybetween 200 nM to 10 μM.

Therefore, suitable amounts of contemplated compounds will be in therange of 0.1 μg per dosage unit to about 0.5 gram per dosage unit, moretypically between 10 μg per dosage unit to about 0.05 gram per dosageunit, and most typically between 50 μg per dosage unit to about 100 mgper dosage unit. Thus, suitable dosages will be in the range of about0.01 μg/kg and 100 mg/kg, more typically between 1 μg/kg and 50 mg/kg,and most typically between 10 μg/kg and 10 mg/kg.

With respect to dosage units, it is generally contemplated that thedosage unit will be such that the dosage unit is effective to achievethe desired therapeutic and/or prophylactic effect. Viewed from adifferent perspective, a dosage unit will preferably contain sufficientquantities of the calcium flux agonist to (preferably synergistically)increase IL-8 production and/or NF-κB signaling.

It should further be appreciated that while contemplated compounds andcompositions may be applied topically or in a pharmaceutical composition(e.g., cream, ointment, etc.) numerous alternative methods ofapplication to the affected tissue are also deemed suitable. Forexample, contemplated compositions may be coupled to or incorporatedinto a carrier that is directly and reversibly applied to the site oftreatment or that is implanted or otherwise placed in proximity orcontact with the treatment site. For example, contemplated compounds andcompositions may be incorporated into one or more portions of topicallyapplied and removable carriers (e.g., bandages, gauze, etc.) or intocovering films that may or may not dissolve or erode (e.g., viabiodegradable drug-eluting polymers). Alternative carriers include beadsor biodegradable drug-eluting polymers that are implanted whereincontemplated compounds and compositions may be part of the surface ofthe implanted device or coated onto such devices.

Dose Response of Calcium Flux Agonists

The inventors performed several studies to identify the effect ofcalcium flux agonists on various components of an immune response, andparticularly on the effect of calcium flux agonists on IL-8 productionand activation of NF-κB signaling. As can be readily taken from FIGS.1A-1C, both ionophores and SERCA inhibitor produced significantincreases in IL-8 production and activation of NF-κB signaling. The doseresponse is observed over relatively large windows for ionophores (e.g.,A23187 and ionomycin) and over at least two orders of magnitude forSERCA inhibitors (e.g., thapsigargin).

More specifically, the dose response of ionophores on NF-κB and IL-8 isshown in FIGS. 1A-1B using a monocyte-derived cell line to treatment andA23187 and ionomycin in the absence of externally added TLR or NODligands. A human monocytic cell line (THP-1) transfectant possessing astably integrated NF-κB/luciferase reporter cassette was treated withincreasing concentrations of A23187 and ionomycin and NF-κBreporter-based responses were monitored using luminometry (RLU=relativelight units, filled circles) while human IL-8 (hIL-8) release wasquantified using a sandwich ELISA (empty circles). FIG. 1C depicts thedose response to thapsigargin on NF-κB and IL-8. Here, the humanmonocytic cell line (THP-1) transfectant possessing a stably integratedNF-κB/luciferase reporter cassette was treated with increasingconcentrations of thapsigargin and NF-κB reporter-based responses weremonitored using luminometry (RLU=relative light units, filled circles)while human IL-8 (hIL-8) release was quantified using a sandwich ELISA(empty circles). As can be taken from both flux agonist groups, doseresponse is increasing over orders of magnitude at significantlyincreasing NF-κB and IL-8 responses.

Human IL-8 activity: A titration of thapsigargin (A.G. Scientific, SanDiego Calif.), ionomycin (Sigma-Aldrich, St. Louis Mo.), and the calciumionophore A23187 (Sigma-Aldrich) was added to a 96-well plate (ThermoMatrix, Waltham Mass.) seeded with 150,000 THP-1 cells (ATCC TIB-202)grown in RPMI 1640 (Cellgro, Herndon Va.) supplemented with 10% FBS(Thermo HyClonc, Waltham Mass.) and 1% penicillin-streptomycin-fungizone(Thermo HyClone). 24 hours after addition, 25 μL of supernatant wasremoved and assayed for human IL-8 using the Meso Scale Discovery HumanIL-8 Tissue Culture Kit (MSD, Rockville, Md.) per manufacturer'sprotocol. The results were analyzed using the Meso Scale DiscoverySECTOR Imager 2400 and normalized to a titration of human IL-8 suppliedby the MSD Kit.

NF-κB activation: A titration of thapsigargin (A.G. Scientific),ionomycin, and the calcium ionophore A23187 was added to a 96-well plate(Thermo Matrix) seeded with 150,000 THP-1 κB-LUC THP-1's (THP-1 cellscontaining the stable integration of a NF-κB Luciferase Reporter) grownin RPMI 1640 (Cellgro, Herndon Va.) supplemented with 10% FBS (ThermoHyClone) and 1% penicillin-streptomycin-fungizone (Thermo HyClone). 24hours after addition, 25 μL of Bright-Glo Luciferase Assay System(Promega, Madison Wis.) was added to each well and analyzed forluciferase activity using Perkin Elmer TopCount NXT (Perkin Elmer,Waltham Mass.).

Calcium Flux Agonists as Synergistic Agents with Pattern RecognitionReceptors

In an initial set of experiments, the TLR2 receptor ligand Pam2CYS wastested in the presence of various concentrations of calcium fluxagonists and the inventors unexpectedly found that suboptimal andrelatively low concentrations of a variety of calcium flux agonists inthe presence of a TLR ligand produced a dramatic synergistic response.

For example, FIG. 2 is a graph depicting a typical synergistic effectselected calcium flux agonists with an exemplary TLR ligand. Here, theionophores A23187 and ionomycin, as well as the SERCA inhibitorthapsigargin synergize with the TLR2 ligand (Pam2CYS) in the activationof cytokine release from the human THP-1 promonocytic leukemia cellline. It should be especially noted that while the TLR2 ligand aloneprovided for about 5 ng/ml IL-8 response, addition of a suboptimal doseof the calcium flux agonists (e.g. 20 nM for thapsigargin, 316 nM forA23187, and 1 μM for ionomycin) produced more than 10-fold quantities ofIL-8 production in the cells.

To investigate if that observation was also true for other patternrecognition receptors, and especially for TLR and NOD receptors andother ligands, the inventors tested numerous TLR and NOD receptors andvarious ligands. Notably, as is evidenced from FIGS. 3A-3E, substantialsynergy was observed across a large selection of types and classes ofcalcium flux agonists, as well as various TLR and NOD receptors andligands, thus establishing that TLR- and NOD-mediated signals can be(typically synergistically) enhanced with suboptimal dosages of calciumflux agonists.

More specifically, FIG. 3A shows graphs depicting the synergistic effectof suboptimal doses of A23187 to augment cellular response for toll-likereceptor (TLR) family members (TLR2, TLR4, TLR5, TLR9), and NOD familymembers (NOD1, NOD2). FIG. 3B shows graphs depicting the synergisticeffect of suboptimal doses of ionomycin to augment cellular response fortoll-like receptor (TLR) family members (TLR2, TLR4, TLR5, TLR9), andNOD family members (NOD1, NOD2). FIG. 3C shows graphs depicting thesynergistic effect of suboptimal doses of cyclopiazonic acid to augmentcellular response for toll-like receptor (TLR) family members (TLR2,TLR4, TLR5), and NOD family members (NOD1, NOD2). FIG. 3D shows graphsdepicting the synergistic effect of suboptimal doses of2,5-di(tert-butyl) hydroquinone (DBHQ) to augment cellular response fortoll-like receptor (TLR) family members (TLR2, TLR4, TLR5), and NODfamily members (NOD1, NOD2). FIG. 3E shows graphs depicting thesynergistic effect of suboptimal doses of thapsigargin to augmentcellular response for toll-like receptor (TLR) family members (TLR2,TLR4, TLR5, TLR9), and NOD family members (NOD1, NOD2). The doses usedin this experiment were 20 nM for thapsigargin, 316 nM for A23187, 1 μMfor ionomycin, 10 μM for cyclopiazonic acid, and 31.6 μM for DBHQ.

As can be readily appreciated, the synergistic effect with respect toIL-8 production was observed for all of the tested TLR and NOD familymembers and suitable ligands, while all of the tested calcium fluxagonists were used at suboptimal concentration. Thus, a clear pattern ofadjuvant activity of calcium flux agonists on the immune response, andespecially on IL-8 production and NF-κB signaling is evident where theimmune response is associated with a TLR- or NOD-mediated event.

Nature of Calcium Flux

The inventors further investigated if the nature of calcium flux was ofsignificance as increased intracellular calcium may have differentorigins. To that effect, the inventors used various calcium signalingmodulators to investigate the nature of the calcium flux andcontribution. As can be readily taken from the data shown, extracellularcalcium flux into the cell was critical for synergistic amplification ofcalcium signaling. More specifically, FIG. 4 illustrates the effects ofdifferent Ca²⁺ modulating compounds on synergy with respect to NF-κBactivation. It should be noted that EGTA is an extracellular chelator,and that KN-62 is an inhibitor of Cam Kinase II (important for T-cellactivation) that does not have chelator effect. As can be readilyappreciated from FIG. 4, the extracellular Ca²⁺ scavenger EGTA reducessynergistic signal down to levels in line with what is achieved by TLRligand alone while treatment with the Cam Kinase II inhibitor KN-62 doesnot alter reporter gene expression levels.

Previously, it was demonstrated that TLR2 and NOD2 play important rolesin the sensing and control of S. aureus infections by mammalian cells.Given that THP-1-derived κB-LUC THP-1 cells were activated by thegeneric TLR2 and NOD2 ligands Pam2Cys and MDP, respectively, and thatthis activation could be further augmented through the addition ofcalcium flux agonists (FIGS. 3A-3E), the ability of these cells torecognize bona fide S. aureus-derived products was evaluated as follows.Briefly, a culture of logarithmically dividing S. aureus was washedthree times with RPMI 1640 (Cellgro). Concurrently, THP-1 κB-LUC THP-1's(THP-1 cells containing the stable integration of a NF-κB luciferasereporter gene) cultured in RPMI 1640 (Cellgro) supplemented with 10% FBS(Thermo HyClone, Waltham Mass.) and 1% penicillin-streptomycin-fungizone(Thermo HyClone) were washed three times with RPMI 1640 (Cellgro) andtransferred to the bottom compartment of a 24-well 0.4 μm cutofftranswell plate (Corning) at a concentration of 1.2 million/mL.Dilutions of the washed S. aureus culture were then added to the topchamber of the transwell system at the indicated initial multiplicitiesof infection (MOI). The cells were incubated for 18 hours at 37° C. atwhich time aliquots of 60,000 cells were transferred to wells in a384-well white bottom plate (Thermo). To evaluate luciferase geneexpression, Bright-Glo Luciferase Assay Reagent (Promega) was added toeach well and the plates were analyzed for luciferase activity using thePerkin Elmer TopCount N×T system (Perkin Elmer). Consistent with theprevious results, the κB-LUC THP-1 reporter cells demonstrate aMOI-dependent increase in reporter activity when co-cultured with livebacteria in an culture system which allows for passive diffusion ofbacterial products but prevents direct contact between whole S. aureusbacteria and the κB-LUC THP-1 reporter cells (FIG. 5A).

To determine whether the recognition of S. aureus-produced productscould be further augmented through modulation of calcium flux, κB-LUCTHP-1 reporter cells were incubated in the presence of a 1:100 dilution(V/V) of sterile filtered S. aureus conditioned (i.e. spent) or unusedsterile media and treated with a suboptimal dose (20 nM) of thapsigarginor dimethyl sulfoxide (DMSO) vehicle control as described above.Briefly, conditioned media was produced by culturing S. aureus in RPMIwith 10% fetal bovine serum overnight in a shaking incubator at 37° C.following which the bacteria were pelleted by centrifugation and thesupernatants filter-sterilized using a 0.2 μm filter (VWR Radnor Pa.).The κB-LUC THP-1 cells were incubated in the presence of theconditioned/spent media for 18 hours prior to luciferase assay asdescribed above. Interestingly, the response of the κB-LUC THP-1reporter cells to the conditioned/spent bacterial culture wassignificantly enhanced in the presence of thapsigargin (FIG. 5B).

To determine whether the observed enhancement in activation of κB-LUCTHP-1 cells by bacterial products achieved by suboptimal thapsigargintreatment was also true for the calcium ionophores A23187 and ionomycin,the experiment was repeated using these ionophores at the suboptimaldoses of 316 nM and 1 μM, respectively. In all three cases, recognitionof S. aureus conditioned/spent media was significantly enhanced in thepresence of any of the calcium flux agonists (FIGS. 5C-5E). Perhaps ofequal interest, the calcium flux-induced synergy could be inhibited bythe addition of the extracellular chelator EGTA (1 mM) as can be readilyseen form the Figures.

Role of Extracellular Calcium in Human Cells

To investigate whether the response in human cells is affected by thenature of the calcium flux, the inventors tested primary human monocytesand granulocytes infected with S. aureus. As can be seen from the datain FIG. 6, there is a clear requirement of human primary monocytes andgranulocytes for extracellular Ca²⁺ for optimal killing of phagocytosedS. aureus. Briefly, primary human monocytes were purified from acommercially available peripheral blood mononuclear cell preparation(Human Buffy Coat Leukocytes, Innovation Research, Novi Mich.) using theDynabeads Untouched Human Monocyte kit (Life Technologies, Grand Island,N.Y.) per manufacturer's suggestions. Primary human granulocytes werepurified by separating normal donor blood into two fractions byFicoll-Paque density centrifugation by manufacturer's directions (GEHealthcare Life Sciences, Piscataway, N.J.). The peripheral bloodmononuclear cell fraction was then discarded and theerythrocyte/granulocyte fraction was subjected to Red Blood Cell LysisBuffer treatment per manufacturer's protocol (Biolegend, San DiegoCalif.) followed by two washes in Hank's Buffered Salt Solution(Cellgro) to remove the lysis reagent and cell debris. Next, a cultureof S. aureus was grown overnight in tryptic soy broth (Cellgro). Theresulting culture was used to inoculate a new culture to ensure logphase growth prior to the start of the experiment. The bacterial culturewas pelleted (2000×g for 10 min) and washed three times with Hank'sBuffered Salt Solution (Cellgro). Primary monocytes/granulocytesprepared as above were then supplemented with 10% human serum(Innovative Research, Novi Mich.) and 1 mM EGTA (Sigma-Adrich, St. LouisMo.) where applicable and chilled on ice for 30 minutes. After the 30minutes, S. aureus (MOI of 3.33) was added and the mixtures wereincubated on ice for an additional 20 minutes to synchronize binding.Tubes were then incubated for 20 minutes at 37° C. After 20 minutes, 10U/mL of lysostaphin (Sigma-Aldrich) was added to each tube to eliminatenon-internalized S. aureus. At the desired time points (20, 30, 60minutes) 20 uL aliquots were removed and diluted in 10 mL of water. Thedilution tubes were vortex and allowed to sit at room temp for 10minutes to ensure proper lysis of the primary cells. After 10 minutes,the dilution tubes were centrifuged for 10 minutes at 2000×g with theremaining S. aureus was concentrated 20 fold. The S. aureus-containingsamples were then plated in triplicate on tryptic soy agar (Cellgro) andincubated overnight at 37° C. to allow colony enumeration the followingday.

In Vivo Models on Infection and Wound Healing

As already noted above, calcium flux agonists synergize with TLR and NODligands to activate pathways relevant to immune activation. Given theprevalence of TLR and NOD ligands in infection and injury, the inventorstherefore contemplate that introduction of calcium flux agonists inthese contexts will greatly decrease the duration of disease and/oraugment repair function. To substantiate such model, various calciumflux agonists were tested in a pre-infection treatment experiment intheir ability to alter wound healing kinetics and impact bacterialclearance.

To determine if calcium flux agonists such as the ionophores A23187 andionomycin or the SERCA pump inhibitor thapsigargin can be used as apre-treatment of bacterial infection, the inventors treated the shaveddorsal skin of 6-8 week old male C57Bl/6 mice with vehicle alone, orformulations containing 2 mM A23187, ionomycin, or thapsigargin. One daylater, the inventors superficially infected the dorsal skin of groups of5 mice with 2×10⁶ CFU of S. aureus and evaluated bacterial burden andwound size over the next week. Using this model, the inventorsinvestigated if the calcium flux agonists would have a beneficial effecton wound healing (as measured by size) as demonstrated in FIG. 7.Interestingly, animals treated with either ionophore or thapsigargindemonstrated significantly smaller wound sizes (p<0.01 as determined byStudent's T-test) on days 3, 5 and 7 of infection as compared to controltreated animals. Furthermore, animals pre-treated with formulationscontaining either ionophorc or thapsigargin possess significantly fewerbacteria at the infection site on day 7 compared to vehicle controls(FIG. 8).

Topical delivery: A23187 and ionomycin and thapsigargin werereconstituted in dehydrated ethanol (Spectrum Chemicals, Gardena Calif.)to appropriate concentrations for subsequent formulations. In thepre-infection in vivo study, topical formulations consisted of 75%dehydrated ethanol (Spectrum Chemicals), 22% cyclohexane(Sigma-Aldrich), and 3% dimethyl sulfoxide (Sigma-Aldrich). Each topicalformulation was delivered by submersing a circular piece of Whatmanpaper (Whatman, a division of GE Healthcare) with a 1.0 centimeterdiameter and applying said circle to the skin of each recipient for 5minutes.

Preparation of S. aureus for skin inoculation: Briefly, mid-logarithmicphase S. aureus bacteria was washed twice and resuspended in sterilesaline (0.9%) at the noted concentrations. Mice: Male mice, 6-8 weeksold, on a C57BL/6 genetic background were used in all experiments(Jackson Laboratories, Bar Harbor, Me.).

Mouse model of S. aureus skin wound infection: To prepare animals forwound infection, the skin of the posterior upper back and neck of micewas shaved using #40 clippers. Next, three parallel 8 mm longfull-thickness scalpel cuts (no. 11 blade) were made into the dermis.Resulting wounds were inoculated with 10 μl of S. aureus (2×10⁸ CFUs permL) with a micropipettor. Total lesion size (cm2) measurements werequantified by determining total pixel count from photographed animalsusing a millimeter ruler as a reference.

Quantification of in vivo S. aureus bacterial burden: To determine invivo bacterial burden, infected mice were sacrificed on day 7 followinginfection and lesions were harvested surgically. Harvested tissues werehomogenized and bacterial count was determined following plating onappropriate solid growth media.

Antibacterial Effect

Consistent with these and previous results regarding their inherentantibiotic activity against S. aureus, the inventors confirmed thedirect antibiotic activity of both ionophores for direct antibioticactivity on methicillin-sensitive S. aureus (MSSA). As would bepredicted, both ionophores were active against MSSA in a liquid cultureassay with A23187 being the more active compound. Interestingly, bothcompounds demonstrated significant antibiotic activity against amethicillin-resistant S. aureus (MRSA) strain.

For example, FIGS. 9A and 9B are graphs showing the antibioticsensitivity of S. aureus against A23187 and ionomycin, respectively, andFIG. 9C shows antibiotic sensitivity of S. aureus against CPA. As can betaken from the graphs, both ionophores had some direct antibioticeffect, however, that effect was substantially less than the controlusing kanamycin as direct antibiotic. In contrast, FIG. 10 is a graphdepicting lack of direct antibiotic effect of the SERCA inhibitorthapsigargin against S. aureus, even at high concentrations. Todetermine the antibiotic sensitivity of S. aureus, cultures were grownovernight in tryptic soy broth (Cellgro). The resulting cultures wereused to inoculate new cultures to ensure log phase growth prior to thestart of the experiment. Once the bacteria had reached log phase thebacteria was diluted to the appropriate OD₆₀₀˜0.05 and transferred to a24-well plate. Compounds of interest were added and plates wereincubated in a shaking incubator at 37° C. Aliquots were removed atvarious time points and their respective OD₆₀₀ was measured using aBioTek Synergy2 microplate reader (BioTek, Winooski, Vt.). For wellswhose turbidity was affected by the addition of the compounds ofinterest, aliquots were removed and plated on tryptic soy agar (Cellgro)and incubated overnight in a 37° C. incubated and counted the followingday. Thus, it should be appreciated that the calcium flux agonists areeffective in antimicrobial treatments via an indirect effect, mostlikely due to the enhanced production of IL-8 and increased NF-κB-driventranscription.

Moreover, the inventors also discovered that the indirect antibioticeffect can be used in a prophylactic manner. For example, the inventorsdemonstrate that the promonocytic THP-1 cells treated thapsigargindisplayed greater antimicrobial activity than similarly matured cellsnot treated with thapsigargin (FIG. 11). Briefly, a logarithmicallydividing S. aureus was pelleted (2000×g for 10 min) and washed threetimes with Hank's Buffered Salt Solution (Cellgro). Concurrently, 2 daydifferentiated THP-1 cells (using 1 uM retinoic acid and 1 uMcholecalciferaol, Sigma-Aldrich) followed by 1 day with 20 nMthapsigargin or vehicle were collected and washed in Hank's BufferedSalt Solution. Differentiated cells were supplemented with 10% humanserum (Innovative Research) and chilled on ice for 30 minutes. After the30 minutes, S. aureus (MOI of 10) was added on ice for an additional 20minutes to synchronize binding. Tubes were then incubated for 20 minutesat 37° C. After 20 minutes, 10U/mL of lysostaphin (Sigma-Aldrich) wasadded to eliminate non-internalized bacteria. At the desired time points(20, 30, 60 minutes) 20 uL aliquots were removed and diluted into 10 mLof water. The dilution tubes were vortex and allowed to sit at room tempfor 10 minutes to ensure proper lysis of the primary cells. After 10minutes, the dilution tubes were centrifuged for 10 minutes at 2000×gand the remaining S. aureus was concentrated 20 fold and plated intriplicate on tryptic soy agar (Cellgro) and incubated overnight at 37°C. prior to colony enumeration.

Consequently, it should be appreciated that one or more ionophores canbe employed as topical prophylactic and/or therapeutic agents fortreatment of skin infections and/or to improve wound healing. Ofparticular significance is the use of such compositions and methods inan immunostimulatory manner rather than in a direct antibiotic manner,which will advantageously avoids difficulties otherwise associated withresistance build-up due to antibiotic therapy. Of course, it should beappreciated that numerous other pathogens are also contemplated herein,and in fact include all currently known skin pathogens (bacterial,viral, parasitical, and fungal).

It should still further be appreciated that a significant synergisticeffect was observed during treatment with contemplated compounds, wherethe synergy was between the ionophores and TLR ligands in activatingimmune cells. Such synergy could be of particular interest fortreatments that are already directed to modification of immune response(e.g., drug therapy using Aldara) or other immune-activating therapiesto combat disease. Therefore, the inventors contemplate that ionophorescan be used therapeutically to combat superficial skin infections beforeand after onset. Moreover, and given the dual physical barrier andimmunological functions of the skin and the wealth of in vitro datademonstrating the activating/modulating activities of the ionophores inimmune cells, the inventors also contemplate that topical formulationscontaining ionophores will be of therapeutic benefit in patientsrequiring treatment for acute (e.g., from injury) or chronic (e.g., asobserved in diabetic ulcers, etc) superficial wounds, burns, and otherinflammatory/autoimmune disorders of the skin (e.g., psoriasis, eczema,etc).

Besides the skin, epithelial tissues include the cells lining thegastrointestinal tract (including the alimentary cavity), therespiratory tract and the urogenital tract. Other than the latter, theremaining tissues are constantly exposed to microorganisms and otherfactors such as pollen and man-made environmental pollutants, which cancause or otherwise exacerbate local inflammation and/or lesionformation. Due to the prevalence of microorganisms which activate TLRreceptors throughout the gastrointestinal tract (including the mouth),it is also contemplated to utilize ionophores in the treatment of mouthabscesses and possibly inflammatory bowel disorders.

Consequently, it should be appreciated that topically-appliedthapsigargin can be used as a treatment agent to synergize with TLRligands in activating immune cells. Most notably, the inventorsdiscovered that skin treated with thapsigargin or other calcium fluxagonists prior to infection clears live bacteria and heals faster thancontrol-treated skin, Of equal importance, the inventors showed that thedifferences observed in vivo are unlikely to be due to inherentantibiotic property of thapsigargin. As a result, the inventorscontemplate that thapsigargin or other calcium flux agonists can be usedtherapeutically to combat superficial skin infections caused bypathogens that produce or contain TLR/NOD ligands (e.g., S. aureus)after their onset. Given the dual physical barrier and immunologicalfunctions of the skin and the wealth of in vitro data demonstrating theactivating/modulating activities of thapsigargin or other calcium fluxagonists in immune cells, the inventors contemplate that topicalformulations containing thapsigargin are of therapeutic benefit inpatients requiring treatment for acute (e.g., from injury) or chronic(e.g., as observed in diabetic ulcers, etc.) superficial wounds, burns,and other inflammatory/autoimmune disorders of the skin (e.g.,psoriasis, eczema, etc).

Thus, specific embodiments and applications of calcium flux agonistshave been disclosed. It should be apparent, however, to those skilled inthe art that many more modifications besides those already described arepossible without departing from the inventive concepts herein. Theinventive subject matter, therefore, is not to be restricted except inthe spirit of the appended claims. Moreover, in interpreting both thespecification and the claims, all terms should be interpreted in thebroadest possible manner consistent with the context. In particular, theterms “comprises” and “comprising” should be interpreted as referring toelements, components, or steps in a non-exclusive manner, indicatingthat the referenced elements, components, or steps may be present, orutilized, or combined with other elements, components, or steps that arenot expressly referenced.

What is claimed is:
 1. A method of enhancing an immune response in skinof a topically applied medicament, comprising a step of including acalcium flux agonist in the medicament at a concentration effective toenhance the immune response in skin, wherein the immune response isassociated with binding of a ligand to a pattern recognition receptor inan immune competent cell.
 2. The method of claim 1 wherein the patternrecognition receptor is a TLR receptor or a NOD receptor.
 3. The methodof claim 1 wherein the calcium flux agonist is a calcium ionophore. 4.The method of claim 3 wherein the calcium ionophore is ionomycin,calcimycin, calcium ionophore II, calcium ionophore IV, calciumionophore V, or calcium ionophore VI.
 5. The method of claim 1 whereinthe calcium flux agonist is a SERCA inhibitor.
 6. The method of claim 5wherein the SERCA inhibitor is 2,5-Di-tert-butylhydroquinone,thapsigargin, ruthenium red, gingerol, paxilline, or cyclopiazonic acid.7. The method of claim 1 wherein the calcium flux agonist is present inthe medicament at a concentration such that the calcium flux agonist ispresent in the cell in the presence of the ligand at a suboptimalconcentration with respect to a maximum effect of the calcium fluxagonist in the absence of the ligand.
 8. The method of claim 1 whereinthe enhanced immune response is increased IL-8 secretion or increasedactivation of NF-κB signaling.
 9. The method of claim 1 wherein theimmune competent cell is a cell residing in an epidermis or a dermis.10. The method of claim 1 wherein the ligand of the pattern recognitionreceptor is a PAMP.
 11. The method of claim 1 wherein the immuneresponse is enhanced in a synergistic manner in the presence of theligand, and wherein the calcium flux agonist is used in the presence ofthe ligand at a suboptimal concentration with respect to a maximumeffect of the calcium flux agonist in the absence of the ligand.
 12. Themethod of claim 1 wherein the skin is a wounded skin.
 13. The method ofclaim 1 wherein the skin is a wounded skin infected with a bacterialpathogen.